Flow cytometric analysis of THP-1 cells (blue) and K562 cells (green) using Bcl-xL (54H6) Rabbit mAb (Alexa Fluor® 647 Conjugate) (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP®Isotype Control (PE Conjugate) #5742 (dashed lines).
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This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 647 fluorescent dye and tested in-house for direct flow cytometry analysis in human cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated Bcl-xL (54H6) Rabbit mAb #2764.
Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Do not aliquot the antibody. Protect from light. Do not freeze.
All reagents required for this protocol may be efficiently purchased together in our Intracellular Flow Cytometry Kit (Methanol) #13593, or individually using the catalog numbers listed below.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com/flowdyes for a listing of cellular dyes validated for use in flow cytometry.
NOTE: Adherent cells or tissue should be dissociated and in single-cell suspension prior to fixation.
NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.
NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.
NOTE: Antibodies targeting CD markers or other extracellular proteins may be added prior to fixation if the epitope is disrupted by formaldehyde and/or methanol. The antibodies will remain bound to the target of interest during the fixation and permeabilization process. However, note that some fluorophores (including PE and APC) are damaged by methanol and thus should not be added prior to permeabilization. Conduct a small-scale experiment if you are unsure.
NOTE: Count cells using a hemocytometer or alternative method.
posted July 2009
revised August 2019
Protocol Id: 407
Bcl-xL (54H6) Rabbit mAb (Alexa Fluor® 647 Conjugate) detects endogenous levels of total Bcl-xL protein. The antibody does not cross-react with other Bcl-2 family members.Species Reactivity:
Human, Mouse, Rat, Monkey
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Asp61 of human Bcl-xL.
Bcl-xL prevents apoptosis through two different mechanisms: heterodimerization with an apoptotic protein inhibits its apoptotic effect (1,2) and formation of mitochondrial outer membrane pores help maintain a normal membrane state under stressful conditions (3). Bcl-xL is phosphorylated by JNK following treatment with microtubule-damaging agents such as paclitaxel, vinblastine and nocodazole (4,5).
Explore pathways + proteins related to this product.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Alexa Fluor is a registered trademark of Life Technologies Corporation.